• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.137-148
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• 1999

Purpose of the present study was to find the optimal ovulation induction medicine for the maturation and development of immature oocytes and culture media for 2-cell embryos in the mouse model. ICR female mouse aged 6 to 8 weeks, were stimulated with 5 IU PMSG injection. At 47 to 50 hour post-PMSG injection, ovaries were dissected out and oocytes-cumulus complexes were punctured. The oocyte-cumulus complexes were cultured in media containing various ovulation induction medicine, CC, HMG and Metrodin for 18 hours. Female ICR mice were stimulated with 5 IU PMSG and 48 hours later were injected 5 IU of hCG, then female and male mice were mated. At 48 hour post-hCG injection, oviducts were dissected out and 2-cell embryos were flushed. The 2-cell embryos were cultured in various media, Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatography, Baxter) water, Medicult media, HTF (human tubal fluid) media for 96 hours. The results were as follows. 1. When the oocytes-cumulus complexes were cultured in $10^{-9}{\mu}g/ml{\sim}10^{-8}{\mu}g/ml$ of CC, those were suppressed in meiotic maturation $(28.2{\sim}33.7%)$. Whereas the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$, these were not effected in meiotic maturation $(54.5{\sim}72.7%)$. 2. When the oocytes-cumulus complexes were cultured in $10^{-4}{\mu}g/ml{\sim}10^{-1}{\mu}g/ml$ of Metrodin, those were suppressed in meiotic maturation $(35.7{\sim}41.5%)$. Meanwhile the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-5}{\mu}g/ml$, those were not effected in meiotic maturation $(54.2{\sim}70.3%)$. 3. When the oocytes-cumulus complexes were cultured in $10^{-5}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$ of HMG, those were suppressed in meiotic maturation $(48.2{\sim}50.4%)$. As being cultured in $10^{-7}{\mu}g/ml{\sim}10^{-6}{\mu}g/ml$, increased in meiotic maturation $(75.8{\sim}80.7%)$. 4. When the 2-cell embryos were cultured in Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatograpy, Baxter) water, Medicult media, HTF (human tubal fluid) media, developmental rates to blastocyst and hatching for 96 hour were 50.0%, 45.2%, 71.5% and 95.6%, respectively.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.4
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• pp.709-727
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• 2001

The pattern of programmed cell death(PCD) has been examined during the early developmental period of development in mouse embryos, from embryonic day 4.5(E4.5) to E11.5 Embryos from Balb/c breedings were harvested at various embryonic stages between E4.5 and El1.5. Cell death was analysed by in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining in tissue sections and whole embryos. At the blastocyst stage(E4.5), a very few apoptotic cells were found in the inner cell mass of the blastocyst. In the early egg cylinder stage(35.0-5.5), a few apoptotic cells were detected in the embryonic ectoderm, the embryonic endoerm and the proamniotic cavity. In the advanced egg cylinder stage(E5.5-6.5), TUNEL-posifive cells were observed in the extra-embryonic ectoderm and extra-embryonic endoderm as well as in the embryonic ectoderm, embryonic visceral endoderm and proamniotic cavity. In the streak stage(E6.75-7.75), many TUNEL-positive cells were found in the ectoplacental cone. In contrast, only very few apoptotic cells were found in the chorion and extra-embryonic endoderm in extra-embryonic regions. In intra-embryonic region, a few apoptotic cells were randomly found in the embryonic ectoderm, mesoderm and visceral endoderm. At the early somitogenesis stage(E8.0-8.5), most apoptotic cells were observed in the most cranial portion of neural fold (neural ectoderm and adjacent ectoderm). At the mid somitogenesis stage(39.0-9.5), the otic placode first showed TUNEL-positive at this stage. Small number of TUNEL-positive cells were also first seen around optic placode and branchial arches. Three streams of TUNEL-positive cells were clearly seen in the cranial region at 59.5-9.75. At E10.5, apoptotic cells were localized in the developing eye, the junctional portion of medial nasal, lateral nasal and maxillary processes, the lateral portion of branchial arches, the junction of bilateral mandibular processes, and apical ectodermal ridges of limb buds. At E11.5, apoptotic cells were noticeably decreased in most area, except the developing limbs and several somites in the tail region. In this study, the global temporospatial pattern of PCD throughout early development of mouse embryos was discussed. It may provide the basis for further studies on its role in the morphogenesis of the embryo.

• Development and Reproduction
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• v.2 no.2
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• pp.179-187
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• 1998

The effects of prostaglandins in hatching and implantation have been studied but the results were various, and those are not well known by the embryonic stage. The present study examined the effects of prostaglandin $E_2$(PG $E_2$) and prostaglandin $F_2$$_{\alpha}$ (PG $F_2$$_{\alpha}$) on the expansion and hatching of mouse embryos by embryonic stage. Also we tried to measure the concentration of prostaglandins of morula, expanded, and hatching embryos. In early morula stage embryos, high concentration of PG $E_2$(>100$\mu$M) showed cytotoxicity but PG $F_2$$_{\alpha}$ did not. The hatching was inhibited all groups but not gave negative effects on expansion. In 84 hr and 96 hr stage embryos, the hatching rate was decreased at all treatment groups but not inhibited the expansion. When combine prostaglandin with indomethacin, the hatching rate was increased significantly compared to the prostaglandin-treated groups, and as lower and lower the PG $E_2$ concentration, the hatching rate increased to the control level. The embryonic synthesis of PG $E_2$ increased dramatically but that of PG $F_2$$_{\alpha}$ increased gradually. PG $E_2$ showed cytotoxicity at early stage embryos much than late stage embryos, but PG $F_2$$_{\alpha}$ did not. Hatching was inhibited by the high PG $F_2$$_{\alpha}$ concentration. It is suggested that the inhibition of hatching might be at resulted from cytotoxicity of PG $E_2$ on embryo. However, it is thought that the mechanisms of inhibition of hatching are different between PG $E_2$ and PG $F_2$$_{\alpha}$. In conclusion, it can be suggested that PG $E_2$ and PG $F_2$$_{\alpha}$ concerned with the expansion and hatching, and their effects on hatching were different by the embryonic stage.$/ concerned with the expansion and hatching, and their effects on hatching were different by the embryonic stage.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.111-124
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• 1996

The present study was carried out to investigate the effects of co-culture cells and growth factors on in vitro culture of Korean native cattle(KNC) embryos fertilized in vitro. Two-eight cell embryos were cultured in vitro using 4 types of co-culture cells and 3 growth factors singly or in combination. The results were as follows, In the co-culture of 2~8 cell embryos with bovine oviductal epithelial cell(BOEC), granulosa cell(BGC), uterine epithelial cell(BUEC) and mouse embryonic fibroblast (MEF) monolayers, the developing rate to blastocysts was significantly(P<0.05) higher with BUEC(32.1%) than with MEF(15.3%), BGC(13.2%) and non co-culture control(11.6%). When the morula co-cultured with BOEC for 5 days following in vitro fertilization were co-cultured with BOEC continuously or with BUEC, respectively, the developing rate to blastocysts was higher with BUEC(73.9%) than with BOEC(56.0%). To examine the effects of growth factors on in vitro development of 2~8 cell embryos, epidermal growth factor(EGF), transforming growth factor-$\beta$l(TGF-$\beta$l) and insulin-like growth factor-1(IGF-1) were added singly or in combination to TCM 199 maturation medium with respective concentration. In a addition of each 10, 30 and SOng /rnl EGF, the developing rate to blastocysts was the highest in lOng /ml EGF(25.3%). In addition of each 1, 2 and Sng /mi TGF-$\beta$1, the developing rate to blastocysts was the highest in lng /ml TGF-$\beta$1(28.8%). In addition of each 50, 100ng/ml JGF-l, the developing rate to blastocysts was higher in 100ng/ml IGF-l(16.5%) than in SOng/mi IGF-1(12.9%). When lOng /ml EGF and lng /ml TGF-$\beta$l was added singly or in combination, the developing rate to blastocysts was similar in groups added singly or in combination with EGF and TGF-$\beta$l (23.l~24.6%), although higher than in control(16.7%). In the co-culture of 2~8 cell embryos Wth BOEC + each 10, 30 and 5Ong /rnl EGF, the developing rate to blastocysts was significantly(p<0.05) higher in BOEC + long /ml EGF(32.3%) than in BOEC + 3Ong /ml EGF(18.9%) and BOEC + song /ml EGF(9.7%). In the co-culture of 2~8 cell embryos with BOEC + each 1, 2, Sng /ml TGF-$\beta$l the developing rate to blastocysts was higher in BOEC + Sng/rnl TGF-$\beta$l(28.2%) than in BOEC + lng /ml TGF-$\beta$l(21.7%) and BOEC + 2ng/ml TGF-$\beta$l(21.4%). In summary, higher developing rate to blastocysts were obtained with co-culture of BUEC for co-culture system, with addition of lOng /ml EGF or lng /ml TGF-$\beta$l for growth factor culture system, and with co-culture of BOEC + lOng /ml EGF or BOEC + Sng /ml TGF-$\beta$l for co-culture + growth factor culture system.

• Development and Reproduction
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• v.13 no.4
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• pp.305-312
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• 2009

Multipotent spermatogonial stem cells (mSSCs), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESCs). The aim of this study was to evaluate the potential for transgenesis of mSSC derived from outbred mice and the production of transgenic animal by the mSSC-insertion into embryo. mSSCs, established from outbred mice (ICR strain) in the previous study, were maintained and then transfected with a lenti-viral vector expressing green fluorescent protein (GFP), CS-CDF-CG-PRE. Embryonic stem cells (ESCs) were derived from inbred transgenic mice (C57BL/6-Tg (CAG-EGFP)) and were used as an experimental control. Transfected mSSCs were well proliferated in vitro and maintained their characteristics and normal karyotype. Ten to twelve mSSCs and ESCs were collected and inserted into perivitelline space of 8-cell mouse embryos, and then transferred them into uteri of poster mothers after an additional 2-days of culture. Percentage of mSSC-derived offsprings was 4.8% (47/980) and which was lower than those (11.7% (67/572)) of ESC-derived ones (P<0.05). However, even though different genetic background of mSSC and ESC origin, the production efficiency of coat-colored chimeric offspring in mSSC group was not different when compared it with ESC (6.4% (3/47) vs. 7.5% (5/67)). From these results, we confirmed that mSSC derived from outbred mice has a pluripotency and a potential to produce chimeric embryos or mice when reaggregatation with mSSC is performed.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.4
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• pp.401-408
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• 2017

Salvia plebeia R. Br. (Lamiaceae) has been used in folk medicines in Asian countries, including Korea and China, to treat inflammatory diseases. The focus of our research was on the anti-adipogenic activity of ethanol extract from Salvia plebeia R. Br. (SPE) in 3T3-L1 adipocytes. This study investigated inhibition of differentiation and lipogenesis upon SPE treatment in 3T3-L1 cells. The results reveal that SPE at non-cytotoxic concentration significantly suppressed triglyceride accumulation and reduced expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein-alpha, and sterol regulatory element-binding protein as adipogenic transcription factors in 3T3-L1 adipocytes compared to non-treated control cells. Inducible phosphorylation of AMP-activated protein kinase, acetyl CoA carboxylase, and hormone-sensitive lipase as well as carnitine palmitoyltransferase-1 mRNA expression increased upon SPE treatment, which suppressed expression of fatty acid synthase. In conclusion, these results demonstrate that SPE can inhibit expression of adipogenic genes in 3T3-L1 adipocytes. Our study suggests that SPE has potential anti-obesity effects and is a novel therapeutic functional agent with anti-adipogenic activity via reduction of lipogenesis.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.265-274
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• 1995

가토 20마리의 혈액중 혈소판의 수를 수정 전후로 조사한 결과, 수정전과 비교하여 수정후 1일부터 유의하게 감소하였으며 (26.84%, P<0.01), 이러한 감소는 수정후 5일까지 계속되었다. 수정후 5일동안 혈소판의 감소율과 혈소판의 최저치를 수정란의 수와 비교한 바, 이들간의 유의한 상관관계(r=0.5032)가 있었다. 가토수정란 (2세포기) 각각 30∼40개를 5마리의 비장적출수란가토의 난관에 이식한 다음, 혈소판수의 변화를 조사한 결과, 이식전에 비해 이식후 90분 (P<0.025)과 135∼270분 (P<0.025)에 유의한 감소를 확인하였따. 특히 수정란 이식후 180분경에 가장 크게 감소하였다 (P<0.001). 60∼70개의 2세포기 가토수정란을 24시간 배양한 다음, 배양액을 3마리의 비장적출가토에 주사한 후 혈소판의 변화를 조사하였을 때, 주사후 120분경부터 혈소판의 수가 유의하게 감소하기 시작하였다. 이들 배양액을 동결, 해동한 다음 주사한 실험에서도 유사한 결가를 얻었다. 또한 이 배양액을 비장적출생쥐에 주사한 경우에도 가토에서와 마찬가지의 혈소판 촉진현상이 나타났다. 혈소판 촉진인자(PAF)의 길항제인 kadsurenone이 첨가된 배양액에서 24시간동안 가토수정란을 배양한 다음, 이 배양액을 4마리의 비장적출생쥐에 주사한 결과, 혈소판수의 변화가 일어나지 않았다. 또한 kadsurenone이 첨가된 배양액에서는 2세포기 가토수정란의 상실기와 배반포기까지 발달율은 각각 4와 7%로 대조구의 7과 49%보다 유의하게 낮았다. 따라서 본 연구에서 가토의 초기배는 가토나 생쥐의 혈소판수를 감소시키고, 특히 길항제 처리는 이러한 혈소판 감소현상을 억제시킬 뿐만 아니라 가토 초기배발달을 억제한다는 것을 확인하였다. 결론적으로 가토초기배에서는 PAF 또는 수정란 유래의 PAF가 분비된다는 것을 알 수 있었으며, 이러한 인자는 동결처리에서도 그 기능은 전혀 변하지 않는다고 본다. 이후에 있어서 mouse LIF의 첨가는 돼지의 수정란을 배반포 이후의 단계에까지 발달시킬 수 있었다. 있어서 더 적합한 것으로 판단되었다. 5. 개발된 모형은 논 관개의 물리적 측면과 관리목표 모두를 고려한 것으로 계산된 효율은 벼, 생육 각 단계에서의 효율 비교에 양호한 방법임을 알 수 있다.은 Sharpsburg 점질양토에 대한 S.C.S 한계허용치 10ton/ha/year 이내로 나타났다. 비처리구에서의 토양유실량은 평균 2.56ton/ha/year로 높게 나타난 반면 3개의 서로 다른 추리구인 비수구, 초생수로구 및 Bromegrass구에서는 각각 0.152, 0.192 및 0.290ton/ha/year로 낮은 결과를 가져왔다. 6. 평균 침전량에 대한 L.S.D. 검정 걸과 전시험구중 비처리구가 고도의 유의차를 나타낸 반면 비수구, 초생수로구 및 Bromegrass 목초구 간에는 아무런 유의차가 인정되지 않았다. 7. 농지보전 처리구인 배수구와 초생수로구는 비처리구에 비해 낮은 침두 유출량과 낮은 토양유실량을 나타내었다.구보다 14% 절감되는 것으로 나타났다.작용하는 것으로 사료된다.된다.정량 분석한 결과이다. 시편의 조성은 33.6 at% U, 66.4 at% O의 결과를 얻었다. 산화물 핵연료의 표면 관찰 및 정량 분석 시험시 시편 표면을 전도성 물질로 증착시키지 않고, Silver Paint 에 시편을 접착하는 방법으로도 만족한 시험 결과를 얻을 수 있었다.째, 회복기 중에 일어나는 입자들의 유입은 자기폭풍의 지속시간을 연장시키는 경향을 보이며 큰 자기폭풍일수록 현저했다. 주상에서 관측된 이러한 특성은 서브스톰 확장기 활동이 자기폭풍의 발달과 밀접한 관계가 있음을 시사한다.se that were all low in two aspects, named "the Nonsignificant group". And the issues were high risk perception in general

• Development and Reproduction
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• v.6 no.1
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• pp.55-66
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• 2002

Embryonic stem cells(ES cells) are derived from the inner cell mass(ICM) of blastocysts, which have the potentials to remain undifferentiated, to proliferate indefinitely in vitro, to differentiate into the derivates of three embryonic germ layers. ES cells are an attractive model system for studying the initial developmental decisions and their molecular mechanisms during embryogenesis. Additionally, ES cells of significant interest to those characterizing the various gene functions utilizing transgenic and gene targeting techniques. We investigated the effects of reproductive hormones, gonadotropins(GTH) and steroids on the induction of differentiation and expressions of their receptor genes using the newly established mouse ES cells. We collected the matured blastocysts of inbred mice C57BL/6J after superovulation and co-cultured with mitotically inactivated STO feeder cells. After 5 passages, we confirmed the expression alkaline phosphatase(Alk P) activity and SSEA-1, 3, 4 expressions. The protocol devised for inducing ES differentiation consisted of an aggregation steps, after 5 days as EBs in hormone treatments(FSH, LH, E$_2$, P$_4$, T) that allows complex signaling to occur between the cells and a dissociation step, induced differentiation through attachment culture during 7 days in hormone treatments. Hormone receptors were not increased in dose-dependent manner. All hormone receptors in ES cells treated reproductive hormones were expressed lower than those of undifferentiated ES cell except for LHR expression in E$_2$-treated ES cells group. After hormone induced differentiation, at least some of the cells are not terminally differentiated, as is evident from the expression of Oct-4, a marker of undifferentiated. To assess their differentiation by gene expression, we analyzed the expression of 7 tissue-specific markers from all three germ layers. Most of hormone-treated group increased in the expression of gata-4 and $\alpha$ -fetoprotein, suggesting reproductive hormone allowed or induced differentiation of endoderm.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.227-234
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• 1995

This study was carried out to investigate the effects according to the time course of glucose exposure on the development of one-cell embryos beyond morula in CR$_laa$ medium. One-cell zygotes from B6CBA F$_1$ mice were recovered at 24 ~ 25h after hCG and cumulus cells were removed with 0.1% hyaluronidase. The embryos were pooled and subsequently divided into each groups and cultured in CR$_laa$ at 37$^{\circ}C$ in 5% CO$_2$ in aIr. The embryos were either, placed in CR$_laa$ containing various concentration (5.5, 16.5, 27.5 and 38.5 mM) of glucose for 1 min. and subsequently returned to the fresh culture medium (without glucose), or were transferred to the same media containing glucose at 72 h post hCG. The results obtained in these experiments were summarized as follows: 1. The development rates of zygotes, recovered from the oviducts in M2 and cultured in CR$_laa$ with 3mg/Im FAF-BSA, to expanded blastocysts (25.7%) and hatching bIastocysts (17.6%) were significantly higher than those of zygotes recovered in TL Hepes (0% and 0%, respectively). 2. The development rates of one-cell embryos exposed to 27.5 mM glucose at 72 h post hCG for 1 min, were 68.8% (CR$_1$+BSA) a and 77.1% (CR$_1$+FBS) of expanded blastocyst stage, but there were no significant differences between the embryos exposed for 1 min. or transferred at 72 h. 3. Regardless of glucose concentration (5.5, 16.5, 27.5 & 38.5mM), 45.7~61.5% of embryos developed to the blastocyst stage. There were no significant differences between any of the treatments on the devel-opment of one-cell embryos. Therefore, the detrimental effect of highly concentration was not appeared.